Abstract

Mutations in the human Myh9 gene are linked to well described inherited thrombocytopenias, those associated with abnormally large platelets in autosomal dominant Myh9-related disorders, including the May-Hegglin anomaly. Myh9 encodes nonmuscle myosin heavy chain IIA (NMMHC-IIA), an abundant platelet protein, but cellular mechanisms underlying the platelet production defect remain unknown. In vitro culture of mouse megakaryocytes (MKs) allows one to address these mechanisms. Although NMMHC-IIA localization in blood platelets is structured, its sub-cellular localization in maturing MKs provides few clues about functions in platelet release. Thrombopoietic cultures of mouse hematopoietic progenitors showed no difference in numbers of mature MKs or in efficiency of proplatelet formation in the presence of blebbistatin, a small-molecule inhibitor of NMMHC-IIA ATPase activity. Thus, elaboration of proplatelets appears to occur independent of this activity. We also differentiated Myh9+/− and −/− embryonic stem (ES) cells in vitro. Again, Myh9−/− ES cells expressed the full capacity to produce mature MKs that extend proplatelets. These results suggest that NMMHC-IIA loss does not affect MK maturation and that thrombocytopenia in MYH9-related disorders, which are dominantly inherited, probably does not reflect genetic haploinsufficiency. To determine if mutant NMMHC-IIA forms impose dominant-negative effects or may be toxic toward MKs, we introduced various MYH9 gene forms into primary hematopoietic progenitors and into Myh9+/− and Myh9−/− ES cells. Constructs encoded full-length NMMHC-IIA, R1933X (a mutation commonly encountered in patients), and a tail-region truncation expected to function as a dominant antagonist. Despite adequate expression of the mutant and full-length, wild type forms, we did not observe discernible differences between control, and mutant forms in any cell system. These results imply that myosin IIA function is not required for MKs differentiating in relative isolation in cell culture and that the effects of this gene may not be cell-autonomous. As bone marrow sinusoidal migration of mature MKs is believed to be necessary for proper platelet release, we asked if and how NMMHC-IIA may be involved in this process. Blebbistatin induced a decrease in formation of intracellular actin stress fibres and also enhanced migration of MKs out of trans-well chambers (8 μm pore). Similarly, overexpression of a dominant-negative NMMHC-IIA mutant in mature primary mouse MKs reduced actin stress fibres. Our findings indicate that NMMHC-IIA is dispensable for MK maturation and proplatelet extension, at least in vitro, and likely regulates MK properties that are required for thrombopoiesis in vivo, such as adhesion to other bone marrow elements and cell motility. Ongoing experiments address if NMMCH-IIA can be implicated in MK interaction with the bone marrow microenvironment and in correct timing of MK maturation and platelet release. (ZC and AB made an equal contribution.)

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